Zeba Wunderlich

Shadow enhancers are groups of DNA regulatory elements that control the same target gene and drive overlapping expression patterns. Large-scale surveys have found shadow enhancers control most developmental genes in animal genomes. The way in which shadow enhancers arise and how they subsequently evolve may further illuminate their regulatory logic and mechanisms of action. To investigate the evolutionary origins of shadow enhancers, we searched for sequence signatures of three birth mechanisms: duplication of existing enhancers, transposable element (TE) co-option, and TE-mediated splitting of ancestral regulatory elements in the Drosophila melanogaster and mouse genomes. Using 420 fly shadow enhancer sets and 9,051 mouse shadow enhancer sets, we found detectable duplication evidence in 18.3% of fly shadow enhancer sets and 33.9% of mouse sets. Duplication signatures were more frequent in larger shadow sets, suggesting that repeated duplication can expand regulatory landscapes. TE-derived enhancers were present in both species but were not enriched in shadow enhancers relative to single enhancers, suggesting that TE co-option contributes to enhancer evolution generally rather than preferentially generating redundant enhancer architectures. Finally, TE-mediated enhancer splitting was rare in both genomes. These results indicate that shadow enhancer birth is mechanistically heterogeneous, reflecting a mixture of duplication, TE co-option, and other mechanisms, whose contributions are shaped by genome architecture and evolutionary time. Therefore, we find that overlapping regulatory functions can arise through multiple evolutionary routes and that birth mechanisms can influence, but do not strictly determine, the regulatory logic of the resulting enhancer set.

Developmental processes depend on carefully coordinated gene expression. Expression is modulated by the binding of transcription factors (TFs) to cis-regulatory elements (CREs), like enhancers and promoters. Many computational and experimental approaches have been developed to find CREs, particularly enhancers, in the genome, each with strengths and caveats. Given the increasing availability of ATAC-seq data and methods to find TF binding therein, we hypothesized that we could use TF footprinting tools to find clusters of TF binding events within accessible chromatin that may act as CREs. Using Drosophila anterior-posterior patterning network as a test bed, we used a digital genomic footprinting tool (DGT), TOBIAS, on previously published early embryo ATAC-seq data to characterize the TF footprint landscape of 16 TFs essential for embryonic patterning. Even in this system, with its extensive enhancer annotation, most footprinted TF binding sites lie outside of known enhancers, with intergenic and intronic regions hosting the highest TF footprint count, albeit at low density. To find potential novel enhancers, we identified high-density TF footprint clusters that are highly conserved and overlap with active enhancer histone mark signals. Five high confidence candidates were selected for reporter assay validation and all five were found to drive spatially patterned expression in the embryo. This study shows that even in a highly characterized system, the analysis of footprinted TF binding sites in ATAC-seq data can uncover new regulatory regions and suggests this approach may be helpful in using existing ATAC-seq data to find novel CREs.

To defend against microbes, animals regulate a complex immune response. The Drosophila innate immune system deploys a large transcriptional induction of signaling proteins, antimicrobial effectors, and other critical immune factors. This transcriptional response is encoded in enhancers, cis -regulatory sequences that modulate gene expression by binding transcription factors (TFs). Although enhancers and transcription factor binding sites (TFBSs) have been identified for several immune responsive genes in Drosophila , most enhancers that regulate immune-induced genes are unknown. By identifying enhancers, we can understand how their composition controls expression and contributes to infection outcome. We employ self-transcribing active regulatory-region sequencing (STARR-seq) in a hemocyte-like cell line to identify immune-specific enhancers across the D. melanogaster genome and perform ATAC-seq in hemocytes extracted from adult flies to assess the chromatin state of these enhancers before and after immune stimulus. We identify hundreds of enhancers responsive to IMD stimulation, one of the two primary immune signaling pathways in Drosophila . As expected, immune enhancers are enriched for motifs of Relish, an NF-kB factor, and Kay/Jra, a bZip heterodimer pair, involved in the Imd and JNK pathways respectively, compared with enhancers active in unstimulated cells. However, when grouping enhancers by their target gene's expression timing or functional role or by the enhancers’ chromatin accessibility pre- or post-stimulus, different groups of TFBS motifs are enriched, suggesting distinct regulatory logic for different parts of the immune response. Identification and characterization of the diverse array of enhancers that regulate the innate immune response expand our understanding of how animals fight infections.

To defend against microbes, animals regulate a complex immune response. The Drosophila innate immune system deploys a large transcriptional induction of signaling proteins, anti-microbial effectors, and other critical immune factors. This transcriptional response is encoded in enhancers, cis -regulatory sequences that modulate gene expression by binding transcription factors (TFs). While enhancers and transcription factor binding sites (TFBS) have been identified for several immune responsive genes in Drosophila , most enhancers that regulate immune-induced genes are unknown. By identifying enhancers, we can understand how their composition controls expression and contributes to infection outcome. We employed STARR-seq (Self Transcribing Active Regulatory-Region sequencing) in a hemocyte-like cell line to identify immune-specific enhancers across the D. melanogaster genome and performed ATAC-seq in hemocytes extracted from adult flies to assess the chromatin state of these enhancers before and after immune stimulus. We identified thousands of enhancers responsive to IMD stimulation, one of the two primary immune signaling pathways in Drosophila . As expected, immune enhancers are enriched for motifs of Relish, an NF-κB factor, and Kay/Jra, a bZip heterodimer pair, involved in the Imd and JNK pathways respectively, compared to enhancers active in unstimulated cells. However, when grouping enhancers by their target gene’s expression timing or functional role or by the enhancers’ chromatin accessibility pre- or post-stimulus, different groups of TFBS motifs are enriched, suggesting distinct regulatory logic for different parts of the immune response. Identification and characterization of the diverse array of enhancers that regulate the innate immune response expands our understanding of how animals fight infections.

Conserved NF-κB signaling pathways shape immune responses in animals. In mammals, NF-κB activation patterns and downstream transcription vary with stimulus, cell type, and stochastic differences among identically treated cells. Whether animals without adaptive immunity exhibit similar heterogeneity or rely on distinct immune strategies remains unknown. We engineered Drosophila melanogaster S2* reporter cells as an immune-responsive model to monitor the dynamics of an NF-κB transcription factor, Relish, and downstream transcription in single, living cells. Following immune stimulation, Relish exhibits diverse nuclear localization dynamics that fall into distinct categories, with both the fraction of responsive cells and their activation speed rising with stimulus dose. Pre-stimulus features, including Relish nuclear fraction, predict a cell’s responsiveness to stimulation. Simultaneous measurement of Relish and downstream transcription revealed that the probability of transcriptional bursts from immune-responsive enhancers correlates with Relish nuclear fraction. The number of NF-κB binding sites tunes transcriptional activity among immune enhancers. Our study uncovers heterogeneity in NF-κB activation and target gene expression within Drosophila, illustrating how dynamic NF-κB behavior and enhancer architecture tune gene regulation.

In epithelial tissues, juxtaposition of cells of different phenotypes can trigger cell competition, a process whereby one type of cell drives death and extrusion of another. During growth and homeostasis, cell competition is thought to serve a quality control function, eliminating cells that are “less fit”. Tissues may also attack and eliminate newly arising tumor cells, exploiting mechanisms shared with other instances of cell competition, but that differ, reportedly, in the involvement of the immune system. Whereas immune cells have been shown to play a direct role in killing tumor cells, this has not been observed in other cases of cell competition, suggesting that tissues recognize and handle cancer cells differently. Here, we challenge this view, showing that, in the fruit fly Drosophila, innate immune cells play similar roles in cell killing during classical cell competition as in eliminating tumors. These findings suggest that immune suppression of cancer may exploit the same mechanisms as are involved in promoting phenotypic uniformity among epithelial cells.

Herein, transcription factor Nuclear Factor-kappaB (NF-κB) is characterized as the downstream effector of putative innate immune signaling pathways from two model jellyfish, Aurelia aurita ( Aa ) and Clytia hemisphaerica ( Ch ). Both jellyfish NF-κB proteins consist primarily of the N-terminal DNA-binding/dimerization domain, and they lack the C-terminal ankyrin repeat inhibitory domain found in vertebrate NF-κB proteins. Both jellyfish NF-κB proteins bind to a consensus mammalian NF-κB binding site, and their AlphaFold3-predicted structures on DNA are similar to that of mouse NF-κB p50. Neither Aa - nor Ch -NF-κB activated transcription of an NF-κB-site reporter gene in human cells; however, Aa -NF-κB did activate transcription from a GAL4-site reporter gene in yeast, whereas Ch -NF-κB did not. Aa - and Ch -NF-κB were both constitutively located in the nucleus when expressed in vertebrate cells. Homologs of IκB-like proteins from both species interacted with their corresponding NF-κB proteins in co-immunoprecipitation assays in HEK 293T cells. These IκB-like proteins influenced the subcellular localization of their cognate NF-κB proteins in vertebrate cells, and their ankyrin repeat domains were predicted to interact with Ch -NF-κB in a manner similar to mammalian IκB and NF-κB. RNA-sequencing data from Ch animals indicate that Ch -NF-κB is expressed at high levels in early developmental stages, when Ch -IκB expression is low, suggesting that active Ch -NF-κB controls an early developmental process. In contrast, in adult animals the expression of Ch -IκB is high, suggesting that Ch -NF-κB requires a signal in order to become active. Overall, these results provide comparative information on the structure, activity, and mRNA expression of jellyfish NF-κB pathway proteins among jellyfish, and they suggest roles for NF-κB in developing and adult jellyfish.